This proposal describes a detailed plan for the development of an academic career in experimental pathophysiology. The principal investigator has completed residency and fellowship training in Clinical Pathology and Molecular Genetic Pathology, respectively. The calcium-release activated calcium (CRAC) channel is the paradigmatic store-operated channel (SOC), and calcium influx through CRAC channels is critical for normal immune function. Two essential components of the CRAC channel, the ER Ca2+ sensor STIM1 and the pore-forming subunit Orai1, were identified in 2005 and 2006. Since that time, structure-function studies in overexpression systems have identified regions of both proteins that alter the activation and inactivation of CRAC channels. Despite this progress, there is currently a complete lack of understanding of how these protein domains shape Ca2+ signals and downstream effects in intact cells expressing physiological levels of STIM1 and Orai1. Three such regions, the lysine-rich (K-rich) domain of STIM, the Inactivation Domain of STIM (IDSTIM), and the Calmodulin Binding Domain of Orai (CBDOrai), are absolutely conserved among mammals, suggesting that they have important roles in mammalian physiology. The central hypothesis of this proposal is that the tuning of Ca2+ signals by these protein domains regulates downstream effector function in mammalian cells under physiological conditions. This hypothesis will be tested by studying mutant and WT STIM1 and Orai1 proteins lentivirally expressed at physiological levels in mast cells (MCs) derived from existing STIM1 and Orai1 knockout animals. Specific aims are: (1) Define the effect of the STIM1 lysine-rich domain on MC function; (2) Define the effect of non-inactivating CRAC channels on MC function; (3) Define the effect of hyperinactivating CRAC channels on MC function. These studies will be the first to reveal the effects of changes in CRAC channel gating on cellular Ca2+ dynamics and effector functions in a physiological context, and will advance understanding of both CRAC channel function and MC biology. Drs. Stephen J. Galli and Richard S. Lewis will serve as co-mentors for this project, providing scientific expertise in mast cell physiology and calcium signaling, respectively. An advisory committee with additional expertise will meet regularly to provide scientific feedback and career advice to support the candidate's long-term goal of developing an academic career in experimental pathophysiology. Stanford University Medical Center, and the Galli and Lewis laboratories in particular, offer an environment uniquely suited to completing this project bridging the mast cell and CRAC channel fields, and to facilitating the candidate's development into an independent investigator whose expertise in ion channel biology is complemented by strength in cellular pathophysiology.